目的： 利用小分子干扰RNA(siRNA)技术抑制人直肠癌Colo320细胞c-my基因的表达，探讨c-myc基因在人直肠癌Colo320细胞中的作用。方法： 设计人直肠癌Colo320细胞基因特异性小分子干扰RNA，用体外转录方法合成人直肠癌Colo320细胞的小分子干扰RNA并转染该细胞,培养48～96 h后，收集细胞RNA,应用实时荧光定量PCR方法检测转染细胞中c-myc基因mRNA水平变化,Western blotting检测c-myc表达的蛋白,四甲基偶氮唑蓝(MTT)法和集落形成试验检测细胞增殖活性。结果： 转染siRNA后,与对照组相比，实验组pGensil-c-myc-1、2、3、4的c-myc基因mRNA水平明显降低,c-myc的蛋白表达也明显降低。MTT法和集落形成试验检测表明，实验组的细胞增殖速率明显低于对照组。结论： 在人直肠癌Colo320 细胞中存在RNA干扰的机制,特异性siRNA能够有效地抑制c-myc基因的表达,从而抑制Colo320 细胞的增殖。

Objective: To inhibit c-myc gene expression in human rectal cancer cell line Colo320 by small interfering RNA technique, so as to assess the role of c-myc in Colo320 cells. Methods: The c-myc gene specific siRNA was designed and prepared by in vitro transcription. The prepared c-myc siRNA was transfected into Colo320 cells. After cultured for 48-96 hours, the cells were harvested. c-myc mRNA and protein level was monitored by fluorescence real time reverse transcription-poly merase chain reaction, c-myc protein expression was detected by Western blotting, and the cell proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry and clone test. Results: Compared with control group, the mRNA of pGensil-c-myc-1, 2, 3, and 4 in Colo320 cells was obviously decreased in c-myc siRNA transfected cells. C-myc protein expression was also decreased obviously. MTT assay and clone test showed that c-myc siRNA apparently slowed down the proliferation of Colo320 cells. Conclusion: Our results suggest that RNA interference exists in Colo320 cell line; c-myc siRNA can specifically inhibit the expression of c-myc gene in Colo320 cells, subsequently inhibits the proliferation of Colo320 cells.

湖北省技攻关计划项目(编号2006AA301B66-3)